EP0056629B1 - Process for the purification of blood coagulation factors ii, vii, ix and/or x and preparations manufactured accordingly - Google Patents

Process for the purification of blood coagulation factors ii, vii, ix and/or x and preparations manufactured accordingly Download PDF

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Publication number
EP0056629B1
EP0056629B1 EP82100257A EP82100257A EP0056629B1 EP 0056629 B1 EP0056629 B1 EP 0056629B1 EP 82100257 A EP82100257 A EP 82100257A EP 82100257 A EP82100257 A EP 82100257A EP 0056629 B1 EP0056629 B1 EP 0056629B1
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Prior art keywords
factor
vii
blood coagulation
coagulation factors
adsorbent
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German (de)
French (fr)
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EP0056629A1 (en
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Horst Dr. Schwinn
Norbert Prof. Dr. Heimburger
Gerhardt Kumpe
Heinz Hermann Drescher
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CENTEON PHARMA GMBH;HOECHST AKTIENGESELLSCHAFT
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Behringwerke AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/647—Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a process for the purification of liquids containing the blood coagulation factors 11, VII, IX and / or X by adsorption onto mineral adsorbents described per se such as calcium phosphate, aluminum hydroxide gel, barium sulfate or hydroxyapatite in the presence of calcium ions and elution. It is known to enrich and purify blood coagulation factors by adsorption on anion exchangers and calcium phosphate and elution, for example from plasma (Soulier, J.P. et al.: Thromb. Diath. Haemorrh. Suppl. 35, 61 1969); Bruning, P.F. et al. : Hemophilia and New Hemorrhagic States, Univ. North Carolina Press, Chapel Hill, p. 3 (1979); Bidwell, E. et al. : Br. J. Haematol. 22, 469 (1972).
  • mineral adsorbents described per se
  • these blood coagulation factors are adsorbed and eluted from solutions containing these factors in the presence of calcium ions on mineral adsorbents described per se, such as calcium phosphate, aluminum hydroxide gel, barium sulfate or hydroxyapatite.
  • the invention accordingly relates to a process for the purification of one of the blood coagulation factors II, VII, IX and / or X by adsorption from a solution in the presence of calcium ions onto a mineral adsorbent, characterized in that at a concentration of the calcium ions of 0.05 to 2 mol / I, a concentration of the adsorbent of 0.2 to 20% (w / v) and a pH of 6 to 9 adsorbed, adsorbent and liquid separated, if necessary the adsorbent washed and the blood coagulation factor is eluted.
  • a protein solution containing the blood coagulation factor in a concentration of at least 0.05 to 0.1 units / mg protein is mixed with a calcium salt in a concentration of 0.05 to 2.0 mol / 1, preferably 0.4 to 0, 6 mol / l calcium chloride (CaCl 2 ) and a surface-active adsorbent such as calcium phosphate, aluminum hydroxide gel, hydroxylapatite or barium sulfate in a concentration of 0.2 to 20% w / v, preferably 0.4 to 1% w / v calcium phosphate and stirred at a pH of 6.0 to 9.0.
  • the adsorbent is separated from the liquid, optionally washed and eluted, as in Soulier JP et al., Thromb. Diath. Hemorrhage. Suppl. 35, 61 (1969).
  • the protein solution may have to be fractionated with precipitants known to the person skilled in the art before adsorption, the precipitants to be chosen not changing the concentration of the calcium ions.
  • the method according to the invention is superior to the previously cited methods known from the literature due to the addition of calcium ions during the adsorption.
  • the high affinity of the blood coagulation factors for the adsorbents mentioned in the presence of calcium ions is shown in the following table using the example of 0.5 mol / l CaCl 2 , calcium phosphate and coagulation factor II (prothrombin):
  • factor II it can be, for example, according to the method of Koller, F. et al., Dtsch. med. Weekly 81, 516 (1956). A part, for example 0.1 ml of factor II deficient plasma, and a part are added diluted normal plasma mixed. This mixture is kept at + 37 ° C for 30 seconds. Then you put two parts of calcium-containing thromboplastin, z. B. prepared according to German Patent 23 56 493, and determines the time that elapses before the appearance of a clot. For a quantitative statement, the clotting time resulting from the solution containing factor II is read off with reference to a calibration curve obtained with a normal plasma dilution series.
  • factor II corresponds to the factor II activity of 1 ml of normal plasma.
  • Factor VII can be determined, for example, by the method of Koller, F. et al., Acta haemat. 6.1 (1951). For this purpose, a part, e.g. B. 0.1 ml of factor VII deficient plasma, and a part of diluted normal plasma mixed. This mixture is kept at + 37 ° C for 30 seconds. Then you put 2 parts of calcium-containing thromboplastin, z. B. prepared according to German patent 2356493, and determines the time that elapses before the appearance of a clot. For a quantitative statement, the clotting time resulting from the solution containing factor VII is read off with reference to a calibration curve obtained with a normal plasma dilution series.
  • factor VII corresponds to the factor VII activity of 1 ml of normal plasma.
  • factor IX corresponds to the factor IX activity of 1 ml normal plasma.
  • the factor X can, for example, according to the method of Duckert, F. et al., Blood Coagulation, Hemorrhage and Thrombosis, Ed. Tocantins, L.M. and Kazal, L.A. (1964).
  • a part e.g. B. 0.1 ml, factor X deficiency plasma and a part of diluted normal plasma mixed. This mixture is kept at + 37 ° C for 30 seconds.
  • the clotting time resulting from the solution containing factor X is read off with reference to a calibration curve obtained with a normal plasma dilution series.
  • factor X corresponds to the factor X activity of 1 ml of normal plasma.
  • the process conditions can be controlled from the point of view of a satisfactory yield and a satisfactory purity of a concentrate which may be prepared and contains the blood coagulation factors.
  • the solution is brought to an ammonium sulfate concentration of 40% w / v.
  • the precipitate is centrifuged off and discarded.
  • the supernatant is freed from sulfate ions by dialysis and mixed with 0.25 kg CaCl 2 .2H20 and 0.25 kg Ca 3 (P0 4 ) 2 and stirred at pH 7.6 for 30 minutes.
  • 0.25 kg Ca 3 (P0 4 ) 2 can be used with a comparable result 1.3 l of a 1% w / v Al (OH) 3 suspension.
  • the adsorbent is washed twice with 10 liters of NaCl solution, 0.5 ml / l.
  • the adsorbent is eluted with 1.8 liters of pH 8.0 buffer, the 0.2 mol / l trisodium citrate, 0.15 mol / l NaCl, 2 g / 100 ml glycine, 0.3 U / ml Contains antithrombin III and 14 IU / ml heparin.
  • the eluate is separated from the adsorbent by centrifugation at 30,000 x g.
  • the residue is discarded and the supernatant is dialyzed against 100 liters of a buffer of pH 7 with 0.06 mol / 1 NaCl, 0.02 mol / l trisodium citrate and 2 g / 100 ml glycine for three hours.
  • the dialysate is tested for factor II, VII, IX and X activity, adjusted to the desired concentration, sterile filtered, dosed and lyophilized.
  • Approx. 650 fillings of 160 E factor II, 80 E factor VII, 200 E factor IX and 140 E factor X are obtained from 500 liters of plasma.

Abstract

A process for purifying liquids containing blood clotting factors by adsorption of the factors on mineral adsorbents such as calcium phosphate, aluminum hydroxide gel, barium sulfate or hydroxylapatite in the presence of calcium ions followed by elution is described.

Description

Die Erfindung betrifft ein Verfahren zur Reinigung von die Blutgerinnungsfaktoren 11, VII, IX und/oder X enthaltenden Flüssigkeiten durch Adsorption an an sich hierfür beschriebene mineralische Adsorbentien wie beispielsweise Calciumphosphat, Aluminiumhydroxid-Gel, Bariumsulfat oder Hydroxylapatit in Gegenwart von Calciumionen und Elution. Es ist bekannt, Blutgerinnungsfaktoren durch Adsorption an Anionenaustauscher sowie Calciumphosphat und Elution, beispielsweise aus Plasma, anzureichern und zu reinigen (Soulier, J. P. et al. : Thromb. Diath. Haemorrh. Suppl. 35, 61 1969) ; Bruning, P. F. et al. : Hemophilia and New Hemorrhagic States, Univ. North Carolina Press, Chapel Hill, S. 3 (1979) ; Bidwell, E. et al. : Br. J. Haematol. 22, 469 (1972).The invention relates to a process for the purification of liquids containing the blood coagulation factors 11, VII, IX and / or X by adsorption onto mineral adsorbents described per se such as calcium phosphate, aluminum hydroxide gel, barium sulfate or hydroxyapatite in the presence of calcium ions and elution. It is known to enrich and purify blood coagulation factors by adsorption on anion exchangers and calcium phosphate and elution, for example from plasma (Soulier, J.P. et al.: Thromb. Diath. Haemorrh. Suppl. 35, 61 1969); Bruning, P.F. et al. : Hemophilia and New Hemorrhagic States, Univ. North Carolina Press, Chapel Hill, p. 3 (1979); Bidwell, E. et al. : Br. J. Haematol. 22, 469 (1972).

Aus Hoppe-Seyler's Z. Physiol. Chem., 348 (1967), 1163-1171, besonders Seite 1170, geht hervor, daß Calcium in der Lage ist, die Adsorption von Gerinnungsfaktoren an Bariumsulfat zu hemmen.From Hoppe-Seyler's Z. Physiol. Chem., 348 (1967), 1163-1171, especially page 1170, shows that calcium is able to inhibit the adsorption of coagulation factors on barium sulfate.

Für die Therapie von Gerinnungsstörungen des Blutes werden jedoch Präparationen von Gerinnungsfaktoren von höherer Reinheit gebraucht.However, preparations of coagulation factors of higher purity are needed for the therapy of blood coagulation disorders.

Es bestand daher das Bedürfnis, ein Verfahren zu finden, welches es gestattet, Präparationen der Blutgerinnungsfaktoren II, VII, IX und/oder X aus, gegebenenfalls zur Abtötung von Hepatitisviren erhitzten, Lösungen von der heutzutage geforderten Reinheit zu gewinnen.There was therefore a need to find a process which allows preparations of blood coagulation factors II, VII, IX and / or X to be obtained from solutions of the purity required today, which may have been heated to kill hepatitis viruses.

Diese Aufgabe wird dadurch gelöst, daß diese Blutgerinnungsfaktoren aus diese Faktoren enthaltenden Lösungen in Gegenwart von Calciumionen an an sich hierfür beschriebenen mineralischen Adsorbentien wie beispielsweise Calciumphosphat, Aluminiumhydroxid-Gel, Bariumsulfat oder Hydroxylapatit adsorbiert und eluiert werden.This object is achieved in that these blood coagulation factors are adsorbed and eluted from solutions containing these factors in the presence of calcium ions on mineral adsorbents described per se, such as calcium phosphate, aluminum hydroxide gel, barium sulfate or hydroxyapatite.

Gegenstand der Erfindung ist demnach ein Verfahren zur Reinigung eines der Blutgerinnungsfaktoren II, VII, IX und/oder X durch Adsorption aus einer Lösung in Gegenwart von Calciumionen an ein mineralisches Adsorbens, dadurch gekennzeichnet, daß bei einer Konzentration der Calciumionen von 0,05 bis 2 mol/I, einer Konzentration des Adsorbens von 0,2 bis 20 % (w/v) und einem pH-Wert von 6 bis 9 adsorbiert, Adsorbens und Flüssigkeit getrennt, gegebenenfalls das Adsorbens gewaschen und der Blutgerinnungsfaktor eluiert wird.The invention accordingly relates to a process for the purification of one of the blood coagulation factors II, VII, IX and / or X by adsorption from a solution in the presence of calcium ions onto a mineral adsorbent, characterized in that at a concentration of the calcium ions of 0.05 to 2 mol / I, a concentration of the adsorbent of 0.2 to 20% (w / v) and a pH of 6 to 9 adsorbed, adsorbent and liquid separated, if necessary the adsorbent washed and the blood coagulation factor is eluted.

Eine Proteinlösung, die den Blutgerinnungsfaktor in einer Konzentration von mindestens 0;05 bis 0,1 Einheiten/mg Protein enthält, wird mit einem Calciumsalz in einer Konzentration von 0,05 bis 2,0 mol/1, vorzugsweise 0,4 bis 0,6 mol/I Calciumchlorid (CaCl2) und einem oberflächenaktiven Adsorbens wie Calciumphosphat, Aluminiumhydroxid-Gel, Hydroxylapatit oder Bariumsulfat in einer Konzentration von 0,2 bis 20 % w/v, vorzugsweise 0,4 bis 1 % w/v Calciumphosphat versetzt und bei einem pH-Wert von 6,0 bis 9,0 gerührt. Das Adsorptionsmittel wird von der Flüssigkeit abgetrennt, gegebenenfalls gewaschen und eluiert, wie bei Soulier J. P. et al., Thromb. Diath. Haemorrh. Suppl. 35, 61 (1969) beschrieben.A protein solution containing the blood coagulation factor in a concentration of at least 0.05 to 0.1 units / mg protein is mixed with a calcium salt in a concentration of 0.05 to 2.0 mol / 1, preferably 0.4 to 0, 6 mol / l calcium chloride (CaCl 2 ) and a surface-active adsorbent such as calcium phosphate, aluminum hydroxide gel, hydroxylapatite or barium sulfate in a concentration of 0.2 to 20% w / v, preferably 0.4 to 1% w / v calcium phosphate and stirred at a pH of 6.0 to 9.0. The adsorbent is separated from the liquid, optionally washed and eluted, as in Soulier JP et al., Thromb. Diath. Hemorrhage. Suppl. 35, 61 (1969).

Zum Erzielen einer besseren Ausbeute muß die Proteinlösung gegebenenfalls vor der Adsorption mit Fällungsmitteln, die dem Fachmann bekannt sind, fraktioniert werden, wobei die zu wählenden Fällungsmittel die Konzentration der Calciumionen nicht verändern sollen.To achieve a better yield, the protein solution may have to be fractionated with precipitants known to the person skilled in the art before adsorption, the precipitants to be chosen not changing the concentration of the calcium ions.

Das erfindungsgemäße Verfahren ist durch den Zusatz der Calciumionen während der Adsorption den vorzitierten, literaturbekannten Verfahren überlegen. Die hohe Affinität der Blutgerinnungsfaktoren zu den genannten Adsorbentien in Gegenwart von Calciumionen wird am Beispiel von 0,5 mol/l CaCl2, Calciumphosphat und dem Gerinnungsfaktor II (Prothrombin) in folgender Tabelle gezeigt :

Figure imgb0001
The method according to the invention is superior to the previously cited methods known from the literature due to the addition of calcium ions during the adsorption. The high affinity of the blood coagulation factors for the adsorbents mentioned in the presence of calcium ions is shown in the following table using the example of 0.5 mol / l CaCl 2 , calcium phosphate and coagulation factor II (prothrombin):
Figure imgb0001

Die Gegenwart von CaC12 reduziert die zur quantitativen Adsorption notwendige Calciumphosphat-Konzentration um mehr als das Dreifache. Unspezifische Adsorptionen werden dadurch nahezu ausgeschlossen, so daß die Blutgerinnungsfaktoren nachfolgend in sehr reiner Form eluiert werden können.The presence of CaC1 2 reduces the calcium phosphate concentration required for quantitative adsorption by more than three times. Unspecific adsorption is almost completely excluded, so that the blood coagulation factors can subsequently be eluted in a very pure form.

Die Kontrolle der Maßnahmen zur Anreicherung und Reinigung der an Calciumphosphat, Aluminiumhydroxid-Gel, Bariumsulfat oder Hydroxylapatit adsorbierten Blutgerinnungsfaktoren sind dem Fachmann durch die Kenntnis von Bestimmungsmethoden für die betreffenden Substanzen geläufig.The person skilled in the art is familiar with the control of the measures for the enrichment and purification of the blood coagulation factors adsorbed on calcium phosphate, aluminum hydroxide gel, barium sulfate or hydroxylapatite due to the knowledge of determination methods for the substances in question.

Für Faktor II kann sie zum Beispiel nach der Methode von Koller, F. et al., Dtsch. med. Wochenschr. 81, 516 (1956), erfolgen. Dazu wird ein Teil, zum Beispiel 0,1 ml Faktor-II-Mangelplasma, und ein Teil verdünntes Normalplasma vermischt. Diese Mischung wird 30 Sekunden bei + 37 °C gehalten. Danach setzt man zwei Teile calciumhaltiges Thromboplastin, z. B. hergestellt nach dem deutschen Patent 23 56 493, zu und bestimmt die Zeit, die bis zum Auftreten eines Gerinnsels verstreicht. Zur quantitativen Aussage wird die aus der Faktor II enthaltenden Lösung sich ergebende Gerinnungszeit unter Bezugnahme auf eine mit einer Normalplasma-Verdünnungsreihe erzielten Eichkurve abgelesen.For factor II, it can be, for example, according to the method of Koller, F. et al., Dtsch. med. Weekly 81, 516 (1956). A part, for example 0.1 ml of factor II deficient plasma, and a part are added diluted normal plasma mixed. This mixture is kept at + 37 ° C for 30 seconds. Then you put two parts of calcium-containing thromboplastin, z. B. prepared according to German Patent 23 56 493, and determines the time that elapses before the appearance of a clot. For a quantitative statement, the clotting time resulting from the solution containing factor II is read off with reference to a calibration curve obtained with a normal plasma dilution series.

Eine Einheit Faktor II entspricht der Faktor II-Aktivität von 1 ml Normalplasma.One unit of factor II corresponds to the factor II activity of 1 ml of normal plasma.

Der Faktor VII kann zum Beispiel nach der Methode von Koller, F. et al., Acta haemat. 6,1 (1951) bestimmt werden. Dazu wird ein Teil, z. B. 0,1 ml Faktor VII-Mangelplasma, und ein Teil verdünntes Normalplasma vermischt. Diese Mischung wird 30 Sekunden bei + 37 °C gehalten. Danach setzt man 2 Teile calciumhaltiges Thromboplastin, z. B. hergestellt nach dem deutschen Patent 2356493, zu und bestimmt die Zeit, die bis zum Auftreten eines Gerinnsels verstreicht. Zur quantitativen Aussage wird die aus der Faktor VII enthaltenden Lösung sich ergebende Gerinnungszeit unter Bezugnahme auf eine mit einer Normalplasma-Verdünnungsreihe erzielten Eichkurve abgelesen.Factor VII can be determined, for example, by the method of Koller, F. et al., Acta haemat. 6.1 (1951). For this purpose, a part, e.g. B. 0.1 ml of factor VII deficient plasma, and a part of diluted normal plasma mixed. This mixture is kept at + 37 ° C for 30 seconds. Then you put 2 parts of calcium-containing thromboplastin, z. B. prepared according to German patent 2356493, and determines the time that elapses before the appearance of a clot. For a quantitative statement, the clotting time resulting from the solution containing factor VII is read off with reference to a calibration curve obtained with a normal plasma dilution series.

Eine Einheit Faktor VII entspricht der Faktor VII-Aktivität von 1 ml Normalplasma.One unit of factor VII corresponds to the factor VII activity of 1 ml of normal plasma.

Die Bestimmung des Faktors IX erfolgt beispielsweise nach folgendem Verfahren :

  • 1 Teil, z. B. 0,1 ml partielles Thromboplastin, z. B. hergestellt nach der DE-AS 23 16 430, wird mit einem Teil Faktor IX-Mangelplasma und einem Teil verdünntem Normalplasma vermischt. Diese Mischung wird 6 Minuten bei 37 °C gehalten. Nach Zusatz von einem Teil einer auf 37 °C vorgewärmten 0,025 molaren Calciumchlorid-Lösung wird die Zeit bestimmt, die vom Zusatz der Calciumchlorid-Lösung bis zum Auftreten eines Gerinnsels verstreicht. Zur quantitativen Aussage wird die aus der Faktor IX enthaltenden Lösung sich ergebende Gerinnungszeit unter Bezugnahme auf eine mit einer Normalplasma-Verdünnungsreihe erzielten Eichkurve abgelesen.
Factor IX is determined, for example, using the following procedure:
  • 1 part, e.g. B. 0.1 ml partial thromboplastin, e.g. B. manufactured according to DE-AS 23 16 430, is mixed with a part of factor IX deficiency plasma and a part of diluted normal plasma. This mixture is kept at 37 ° C for 6 minutes. After adding part of a 0.025 molar calcium chloride solution preheated to 37 ° C., the time which elapses from the addition of the calcium chloride solution to the appearance of a clot is determined. For quantitative information, the clotting time resulting from the solution containing factor IX is read with reference to a calibration curve obtained with a normal plasma dilution series.

1 Internationale Einheit (= 1 IE) Faktor IX entspricht der Faktor IX-Aktivität von 1 ml Normalplasma.1 international unit (= 1 IU) factor IX corresponds to the factor IX activity of 1 ml normal plasma.

Der Faktor X kann beispielsweise nach der Methode von Duckert, F. et al., Blood Coagulation, Hemorrhage and Thrombosis, Ed. Tocantins, L. M. and Kazal, L. A. (1964), erfolgen. Dazu wird ein Teil, z. B. 0,1 ml, Faktor X-Mangelplasma und ein Teil verdünntes Normalplasma vermischt. Diese Mischung wird 30 Sekunden bei + 37 °C gehalten. Danach setzt man zwei Teile calciumhaltiges Thromboplastin, z. B. hergestellt nach dem DE-Patent 23 56 493, zu und bestimmt die Zeit, die bis zum Auftreten eines Gerinnsels verstreicht. Zur quantitativen Aussage wird die aus der Faktor X enthaltenden Lösung sich ergebende Gerinnungszeit unter Bezugnahme auf eine mit einer Normalplasma-Verdünnungsreihe erzielten Eichkurve abgelesen.The factor X can, for example, according to the method of Duckert, F. et al., Blood Coagulation, Hemorrhage and Thrombosis, Ed. Tocantins, L.M. and Kazal, L.A. (1964). For this purpose, a part, e.g. B. 0.1 ml, factor X deficiency plasma and a part of diluted normal plasma mixed. This mixture is kept at + 37 ° C for 30 seconds. Then you put two parts of calcium-containing thromboplastin, z. B. prepared according to DE Patent 23 56 493, and determines the time that elapses before the appearance of a clot. For a quantitative statement, the clotting time resulting from the solution containing factor X is read off with reference to a calibration curve obtained with a normal plasma dilution series.

Eine Einheit Faktor X entspricht der Faktor X-Aktivität von 1 ml Normalplasma.One unit of factor X corresponds to the factor X activity of 1 ml of normal plasma.

Unter Verwendung dieser Kontrollmethoden können die Verfahrensbedingungen unter dem Gesichtspunkt einer befriedigenden Ausbeute und einer befriedigenden Reinheit eines gegebenenfalls herzustellenden, die Blutgerinnungsfaktoren enthaltenden Konzentrates gelenkt werden.Using these control methods, the process conditions can be controlled from the point of view of a satisfactory yield and a satisfactory purity of a concentrate which may be prepared and contains the blood coagulation factors.

Unter den erfindungsgemäßen Bedingungen erhält man bei vergleichbarer Ausbeute ein wesentlich reineres Produkt als mit den vorzitierten Verfahren, wie folgende Tabelle zeigt :

Figure imgb0002
Under the conditions according to the invention, a substantially purer product is obtained with a comparable yield than with the previously cited processes, as the following table shows:
Figure imgb0002

Die Erfindung soll an den nachstehenden Beispielen näher erläutert werden :The following examples are intended to illustrate the invention:

Beispiel 1example 1

Herstellung eines Faktor II, VII, IX, X-Konzentrates aus humanem Plasma.Production of a factor II, VII, IX, X concentrate from human plasma.

Nach Soulier, J. P. et al. werden 7,5 Liter einer Lösung hergestellt, in der die Faktoren II, VII, IX und X von 500 Litern Human-Plasma in Gegenwart von 1 mol/I NaCI bei pH 8 enthalten sind. Die spezifische Aktivität der Faktoren II, IX und X muß jeweils - 0,1 Einheiten/mg, des Faktors VII ≥ 0,05 Einheiten betragen.According to Soulier, J.P. et al. 7.5 liters of a solution are prepared in which the factors II, VII, IX and X of 500 liters of human plasma are present in the presence of 1 mol / l NaCl at pH 8. The specific activity of factors II, IX and X must be - 0.1 units / mg, factor VII ≥ 0.05 units.

Die Lösung wird auf eine Ammoniumsulfat-Konzentration von 40 % w/v gebracht. Der Niederschlag wird abzentrifugiert und verworfen. Der Überstand wird durch Dialyse von Sulfationen befreit und mit 0,25 kg CaCl2 · 2H20 und 0,25 kg Ca3(P04)2 versetzt und bei pH 7,6 30 Minuten gerührt. Anstelle von 0,25 kg Ca3 (P04)2 können mit vergleichbarem Ergebnis 1,3 I einer 1 %igen w/v Al(OH)3-Suspension eingesetzt werden.The solution is brought to an ammonium sulfate concentration of 40% w / v. The precipitate is centrifuged off and discarded. The supernatant is freed from sulfate ions by dialysis and mixed with 0.25 kg CaCl 2 .2H20 and 0.25 kg Ca 3 (P0 4 ) 2 and stirred at pH 7.6 for 30 minutes. Instead of 0.25 kg Ca 3 (P0 4 ) 2 can be used with a comparable result 1.3 l of a 1% w / v Al (OH) 3 suspension.

Nach Zentrifugation wird der Überstand verworfen und das Adsorbens mit je 10 Liter NaCI-Lösung, 0,5 ml/l, zweimal gewaschen. Das Adsorbens wird mit 1,8 Liter Puffer von pH 8,0 eluiert, der 0,2 mol/I Tri-Natrium-Citrat, 0,15 mol/I NaCI, 2 g/100 ml Glycin, 0,3 E/ml Antithrombin III und 14 IE/ml Heparin enthält. Nach Zusatz von 0,2 g/100 ml kolloidaler Kieselsäure als Zentrifugationshilfsmittel wird das Eluat durch Zentrifugation bei 30000 x g vom Adsorbens abgetrennt. Der Rückstand wird verworfen und der Überstand gegen 100 Liter eines Puffers von pH 7 mit 0,06 mol/1 NaCI, 0,02 mol/l Tri-Natrium-Citrat und 2 g/100 ml Glycin drei Stunden dialysiert. Das Dialysat wird auf Faktor II, VII, IX und X-Aktivität getestet, auf die gewünschte Konzentration eingestellt, sterilfiltriert, dosiert und lyophilisiert.After centrifugation, the supernatant is discarded and the adsorbent is washed twice with 10 liters of NaCl solution, 0.5 ml / l. The adsorbent is eluted with 1.8 liters of pH 8.0 buffer, the 0.2 mol / l trisodium citrate, 0.15 mol / l NaCl, 2 g / 100 ml glycine, 0.3 U / ml Contains antithrombin III and 14 IU / ml heparin. After adding 0.2 g / 100 ml of colloidal silica as a centrifugation aid, the eluate is separated from the adsorbent by centrifugation at 30,000 x g. The residue is discarded and the supernatant is dialyzed against 100 liters of a buffer of pH 7 with 0.06 mol / 1 NaCl, 0.02 mol / l trisodium citrate and 2 g / 100 ml glycine for three hours. The dialysate is tested for factor II, VII, IX and X activity, adjusted to the desired concentration, sterile filtered, dosed and lyophilized.

Aus 500 Liter Plasma erhält man ca. 650 Abfüllungen zu 160 E Faktor II, 80 E Faktor VII, 200 E Faktor IX und 140 E Faktor X.Approx. 650 fillings of 160 E factor II, 80 E factor VII, 200 E factor IX and 140 E factor X are obtained from 500 liters of plasma.

Claims (1)

  1. A process for purifying one of the blood clotting factors II, VII, IX or X by adsorption from a solution on a mineral adsorbent in the presence of calcium ions which comprises adsorbing at a concentration of calcium ions from 0.05 to 2 mol/I, a concentration of the adsorbent from 0.2 to 20 % w/v and a pH from 6 to 9, separating the adsorbent and the liquid from one another, optionally washing the adsorbent and eluting the blood clotting factor.
EP82100257A 1981-01-21 1982-01-15 Process for the purification of blood coagulation factors ii, vii, ix and/or x and preparations manufactured accordingly Expired EP0056629B1 (en)

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DE19813101752 DE3101752A1 (en) 1981-01-21 1981-01-21 "METHOD FOR PURIFYING THE BLOOD COAGINING FACTORS II, VII, IX AND / OR X AND PREPARATIONS PRODUCED THEREFORE"

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CA1186626A (en) 1985-05-07
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DE3263450D1 (en) 1985-06-20
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MX157315A (en) 1988-11-15
ES508756A0 (en) 1983-04-01
US4411794A (en) 1983-10-25
JPS57139017A (en) 1982-08-27
AU7966182A (en) 1982-07-29
DE3101752A1 (en) 1982-08-26
EP0056629A1 (en) 1982-07-28
ES8305376A1 (en) 1983-04-01
IL64823A (en) 1985-07-31
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